Phosphate tightly associated with protein has been known since the late nineteenth century. Since then, a variety of covalent linkages of phosphate to proteins have been found. The most common involve esterification of phosphate to serine and threonine, with smaller amounts being covalently linked to lysine, arginine, histidine, aspartic acid, glutamic acid, and cysteine. The occurrence of phosphorylated proteins implies the existence of one or more protein kinases capable of phosphorylating amino acid residues on proteins, and also of protein phosphatases capable of hydrolyzing phosphorylated amino acid residues on proteins.
Protein kinases play critical roles in the regulation of biochemical and morphological changes associated with cellular growth and division (D'Urso, G. et al. (1990) Science 250: 786-791; Birchmeier. C. et al. (1993) Bioessays 15: 185-189). They serve as growth factor receptors and signal transducers and have been implicated in cellular transformation and malignancy (Hunter, T. et al. (1992) Cell 70: 375-387; Posada, J. et al. (1992) Mol. Biol. Cell 3: 583-592; Hunter, T. et al. (1994) Cell 79: 573-582). For example, protein kinases have been shown to participate in the transmission of signals from growth-factor receptors (Sturgill, T. W. et al. (1988) Nature 344: 715-718; Gomez, N. et al. (1991) Nature 353: 170-173), control of entry of cells into mitosis (Nurse, P. (1990) Nature 344: 503-508; Maller, J. L. (1991) Curr. Opin. Cell Biol. 3: 269-275) and regulation of actin bundling (Husain-Chishti, A. et al. (1988) Nature 334: 718-721).
The overall level, in cells, of protein tyrosine phosphorylation, as well as the phosphorylated state of any given protein, arises from the balance of Protein Tyrosine Kinase (PTK) and Protein Tyrosine Phosphatase (PTPase) activities. Thus PTPases have been proposed as key regulatory elements of cell growth control (Hunter, 1989, Cell 58:1013-1016).
PTKs were discovered and characterized more than one decade earlier than PTPases and in the last few years a large number of studies has led to the identification of many new PTPases and some of them have been accurately characterized. In addition, findings on the biological role of some PTPases in cells have recently been reported (Pondaven, 1991, Adv Prot Phosphatases 6:35-57). Current work suggests that PTKs and PTPases are equally important in many biological processes ranging from cell growth control to cell differentiation and development. In particular, the ocogenic potential of PTKs and the ability of PTPases to counteract PTK oncogenic activation by antiproliferative action suggests that the genes coding for PTPases, in many instances, may be considered tumor-suppressing genes or even anti-oncogenes The existence of PTPases was first predicted to explain the rapid loss of phosphorylation of in vitro phosphorylated membrane proteins (Carpenter et al., 1979, J Biol Chem 254:4884-4891). The main PTPase in human placenta (PTP1B) was purified to homogeneity and sequenced (Tonks et al., 1988, J Biol Chem 263:6722-2730; Charbonneau et al., 1989, PNAS USA 86:5252-5256). Sequence homology between the catalytic domain of PTP1B and the leukocyte common antigen (LCA, or CD45) was demonstrated, indicating that PTPases can be considered a family of structurally related molecules.
The effects of many growth factors such as NGF, BDNF, NT3, FGF, insulin and IGF1 are known to be mediated by high-affinity receptors with tyrosine kinases activity (Fantl et al. Annu. Rev. Biochem., 62 (1993) 453481; Schlessinger and Ulrich Neuron, 9 (1992) 383-391; Ullrich and Schlessinger Cell, 61 (1990) 203-212). Expression of several tyrosine phosphatase genes has been detected in the brain (Jones et al. J. Biol. Chem., 264 (1989) 7747-7753), including RPTP.alpha. (Kaplan et al. Proc. Natl Acad. Sci. USA, 87 91990) 7000-7004; Sap et al. Proc. Natl Acad Sci. USA, 87 (1990) 6112-6116), RNPTPX (Guan et al. Proc Natl. Acad. Sci. USA, 87 (19910) 1501-1505), STEP (Lombroso et al. Proc. Nat. Acad. Sci. USA, 88 (1991) 7242-7246), SH-PTP2 (Freeman et al. Proc. Natl. Acad. Sci. USA, 89 (1992) 11239-11243), MPTP.delta. (Mizuno et al. Mol. Cell. Biol., 13 (1993) 5513-5523), DPTP99A and DPTP10D (Yang et al. Cell, 67 (1991) 661-673).
Intraventricular administration of either NGF, BDNF, insulin or IGF1 prevents delayed neuronal death in the CA1 subfield of the hippocampus (Beck et al. J. Cereb Blood Flow Metab., 14 (1994) 689-692; Shigeno et al. J. Neurosci., 11 (1991) 2914-2919; Zhu and Auer J. Cereb. Blood Flow Metab., 14 (1994) 237-242).
Tyrosine kinase inhibitors block the tyrosine phosphorylation of MAP kinase (Blenis Proc. Natl. Acad. Sci. USA, 90 (1993) 5889-5892; Pelech and Sanghera Science, 257 (1992) 1335-1356) and prevent delayed neuronal death after forebrain ischemia (Kindy J. Cereb. Blood Flow Metab, 13 (1993) 372-377). During reperfusion after ischemia, tyrosine phosphorylation of proteins increases in the hippocampus but some proteins in the hippocampus are dephosphorylated (Campos-Gonzalez J. Neurochem., 59 (1992) 1955-1958; Hu and Wieloch J. Neurochem, 62 (1994) 1357-1367; Takano et al. J. Cereb. Blood Flow Metab., 15 (1995)33-41). These observations suggest that tyrosine phosphorylation plays an important role in the delayed neuronal death which occurs as a result of ischemia-reperfusion injury.
A number of PTPases, in addition to the hydrolytic activity on phosphotyrosine, show some phosphoserine/phosphothreonine phosphatase activity. These enzymes, mostly localized in the nucleus and referred to as dual-specificity PTPases (dsPTPases), are emerging as a subclass of PTPases acting as important regulators of cell cycle control and mitogenic signal transduction possibly by controlling the activity of signal transduction proteins like ERK. In fact, they appear responsible for in vivo nuclear dephosphorylation and inactivation of nuclear dephosphorylation and inactivation of MAP kinases (Alessi et al., 1995, Curr Biol 5:195-283). These enzymes exhibit sequence identity to the vaccinia H-1 gene product, the first identified dsPTPase (Guan et al., 1991, Nature 350:359-362). Several dsPTPases differing from each other in length have been identified. These enzymes and the other PTPase subclasses share an active site sequence motif showing only a limited sequence homology beyond this region.
Given the importance of such protein tyrosine phosphatases in the regulation of the cell cycle, there exists a need to identify novel protein tyrosine phosphatases which function as modulators in the cell cycle such as the suppression of proliferation and whose aberrant function can result in disorders arising from improper cell cycle regulation such as cancer.